Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now. ¹ ^, ² B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species ³ ^, ⁴ that have often been considered as selfish genetic elements that behave as genome parasites. ⁵ ^, ⁶ The observation that in some species Bs can be either restricted or predominant in one sex ^7-14 raised the interesting hypothesis that Bs could play a role in sex determination. ¹⁵ The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.